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Computer Methods and Programs in... Nov 2023Primary ciliary dyskinesia (PCD) is a rare genetic disorder causing a defective ciliary structure, which predominantly leads to an impaired mucociliary clearance and...
BACKGROUND AND OBJECTIVE
Primary ciliary dyskinesia (PCD) is a rare genetic disorder causing a defective ciliary structure, which predominantly leads to an impaired mucociliary clearance and associated airway disease. As there is currently no single diagnostic gold standard test, PCD is diagnosed by a combination of several methods comprising genetic testing and the examination of the ciliary structure and function. Among the approved diagnostic methods, only high-speed video microscopy (HSVM) allows to directly observe the ciliary motion and therefore, to directly assess ciliary function. In the present work, we present our recently developed freely available open-source software - termed "Cilialyzer", which has been specifically designed to support and facilitate the analysis of the mucociliary activity in respiratory epithelial cells captured by high-speed video microscopy.
METHODS
In its current state, the Cilialyzer software enables clinical PCD analysts to load, preprocess and replay recorded image sequences as well as videos with a feature-rich replaying module facilitating the commonly performed qualitative visual assessment of ciliary function (including the assessment of the ciliary beat pattern). The image processing methods made accessible through an intuitive user interface allow clinical specialists to comfortably compute the ciliary beating frequency (CBF), the activity map and the "frequency correlation length" - an observable getting newly introduced. Furthermore, the Cilialyzer contains a simple-to-use particle tracking interface to determine the mucociliary transport speed.
RESULTS
Cilialyzer is fully written in the Python programming language and freely available under the terms of the MIT license. The proper functioning of the computational analysis methods constituting the Cilialyzer software is demonstrated by using simulated and representative sample data from clinical practice. Additionally, the software was used to analyze high-speed videos showing samples obtained from healthy controls and genetically confirmed PCD cases (DNAI1 and DNAH11 mutations) to show its clinical applicability.
CONCLUSIONS
Cilialyzer serves as a useful clinical tool for PCD analysts and provides new quantitative information awaiting to be clinically evaluated using cohorts of PCD. As Cilialyzer is freely available under the terms of a permissive open-source license, it serves as a ground frame for further development of computational methods aiming at the quantification and automation of the analysis of mucociliary activity captured by HSVM.
Topics: Humans; Respiratory Rate; Software; Programming Languages; Automation; Genetic Testing; Rare Diseases
PubMed: 37598471
DOI: 10.1016/j.cmpb.2023.107744 -
Fungal Biology Dec 2023Unlike the mechanism of ballistospore discharge, which was not solved until the 1980s, the operation of asci as pressurized squirt guns is relatively straightforward and... (Review)
Review
Unlike the mechanism of ballistospore discharge, which was not solved until the 1980s, the operation of asci as pressurized squirt guns is relatively straightforward and was understood in the nineteenth century. Since then, mycologists have sought to understand how structural adaptations to asci have allowed the ascomycetes to expel spores of different shapes and sizes over distances ranging from a few millimeters to tens of centimeters. These modifications include the use of valves at the tips of asci that maintain ascus pressure and expel spores at the highest speeds, and gelatinous appendages that connect spores after release and create larger projectiles with greater momentum than single spores. Clever experiments in the twentieth century coupled with meticulous microscopic studies led investigators to understand how asci with complicated apical structures worked and mathematical models produced estimates of launch speeds. With the recent application of high-speed video microscopy, these inferences about ascus function have been tested by imaging the motion of spores on a microsecond timescale. These experiments have established that ascospore discharge is the fastest fungal movement and is among the fastest movements in biology. Beginning with the history of the study of asci, this review article explains how asci are pressurized, how spores are released, and how far spores travel after their release. We also consider the efficiency of ascospore discharge relative to the mechanism of ballistospore discharge and examine the way that the squirt gun mechanism has limited the morphological diversity of ascomycete fruit bodies.
Topics: Firearms; Ascomycota; Spores, Fungal
PubMed: 38097323
DOI: 10.1016/j.funbio.2023.11.001 -
Journal of Visualized Experiments : JoVE Mar 2021A detailed protocol for preparing small molecule samples for microcrystal electron diffraction (MicroED) experiments is described. MicroED has been developed to solve...
A detailed protocol for preparing small molecule samples for microcrystal electron diffraction (MicroED) experiments is described. MicroED has been developed to solve structures of proteins and small molecules using standard electron cryo-microscopy (cryo-EM) equipment. In this way, small molecules, peptides, soluble proteins, and membrane proteins have recently been determined to high resolutions. Protocols are presented here for preparing grids of small-molecule pharmaceuticals using the drug carbamazepine as an example. Protocols for screening and collecting data are presented. Additional steps in the overall process, such as data integration, structure determination, and refinement are presented elsewhere. The time required to prepare the small-molecule grids is estimated to be less than 30 min.
Topics: Cryoelectron Microscopy; Electrons
PubMed: 33779618
DOI: 10.3791/62313 -
Journal of Biomedical Optics Jun 2024Full-field optical coherence microscopy (FF-OCM) is a prevalent technique for backscattering and phase imaging with epi-detection. Traditional methods have two...
SIGNIFICANCE
Full-field optical coherence microscopy (FF-OCM) is a prevalent technique for backscattering and phase imaging with epi-detection. Traditional methods have two limitations: suboptimal utilization of functional information about the sample and complicated optical design with several moving parts for phase contrast.
AIM
We report an OCM setup capable of generating dynamic intensity, phase, and pseudo-spectroscopic contrast with single-shot full-field video-rate imaging called bichromatic tetraphasic (BiTe) full-field OCM with no moving parts.
APPROACH
BiTe OCM resourcefully uses the phase-shifting properties of anti-reflection (AR) coatings outside the rated bandwidths to create four unique phase shifts, which are detected with two emission filters for spectroscopic contrast.
RESULTS
BiTe OCM overcomes the disadvantages of previous FF-OCM setup techniques by capturing both the intensity and phase profiles without any artifacts or speckle noise for imaging scattering samples in three-dimensional (3D). BiTe OCM also utilizes the raw data effectively to generate three complementary contrasts: intensity, phase, and color. We demonstrate BiTe OCM to observe cellular dynamics, image live, and moving micro-animals in 3D, capture the spectroscopic hemodynamics of scattering tissues along with dynamic intensity and phase profiles, and image the microstructure of fall foliage with two different colors.
CONCLUSIONS
BiTe OCM can maximize the information efficiency of FF-OCM while maintaining overall simplicity in design for quantitative, dynamic, and spectroscopic characterization of biological samples.
Topics: Animals; Microscopy; Tomography, Optical Coherence; Microscopy, Phase-Contrast
PubMed: 38584966
DOI: 10.1117/1.JBO.29.S2.S22704 -
Fly Dec 2021To maintain homoeostasis, cells must degrade damaged or misfolded proteins and synthesize functional replacements. Maintaining a balance between these processes, known... (Review)
Review
To maintain homoeostasis, cells must degrade damaged or misfolded proteins and synthesize functional replacements. Maintaining a balance between these processes, known as protein turnover, is necessary for stress response and cellular adaptation to a changing environment. Damaged mitochondria must also be removed and replaced. Changes in protein and mitochondrial turnover are associated with aging and neurodegenerative disease, making it important to understand how these processes occur and are regulated in cells. To achieve this, reliable assays of turnover must be developed. Several methods exist, including pulse-labelling with radioactive or stable isotopes and strategies making use of fluorescent proteins, each with their own advantages and limitations. Both cell culture and live animals have been used for these studies, in systems ranging from yeast to mammals. In vivo assays are especially useful for connecting turnover to aging and disease. With its short life cycle, suitability for fluorescent imaging, and availability of genetic tools, is particularly well suited for this kind of analysis.
Topics: Aging; Animals; Drosophila Proteins; Drosophila melanogaster; Gene Expression Regulation; Male; Mitochondria
PubMed: 34002678
DOI: 10.1080/19336934.2021.1911286 -
Biology Nov 2020Because of its unique microvascular anatomy, the intestine is particularly vulnerable to microcirculatory disturbances. During inflammation, pathological changes in... (Review)
Review
Because of its unique microvascular anatomy, the intestine is particularly vulnerable to microcirculatory disturbances. During inflammation, pathological changes in blood flow, vessel integrity and capillary density result in impaired tissue oxygenation. In severe cases, these changes can progress to multiorgan failure and possibly death. Microcirculation may be evaluated in superficial tissues in patients using video microscopy devices, but these techniques do not allow the assessment of intestinal microcirculation. The gold standard for the experimental evaluation of intestinal microcirculation is intravital microscopy, a technique that allows for the in vivo examination of many pathophysiological processes including leukocyte-endothelial interactions and capillary blood flow. This review provides an overview of changes in the intestinal microcirculation in various acute and chronic inflammatory conditions. Acute conditions discussed include local infections, severe acute pancreatitis, necrotizing enterocolitis and sepsis. Inflammatory bowel disease and irritable bowel syndrome are included as examples of chronic conditions of the intestine.
PubMed: 33255906
DOI: 10.3390/biology9120418 -
Pediatric Pulmonology Sep 2021Coronavirus 2019 (COVID-19) is typically spread by droplets and has had a remarkable effect on pediatric pulmonology healthcare practice. Here, we aimed to evaluate the... (Observational Study)
Observational Study
INTRODUCTION
Coronavirus 2019 (COVID-19) is typically spread by droplets and has had a remarkable effect on pediatric pulmonology healthcare practice. Here, we aimed to evaluate the effect of the COVID-19 pandemic on the clinical follow-up and hospital visits of patients followed up at a pediatric pulmonology unit at a tertiary care center.
METHODS
This study was an observational descriptive study performed at a tertiary care center pediatric pulmonology unit between January 2019 and December 2020. We analyzed the outpatient visits, laboratory procedures which including pulmonary function tests (PFTs), diffusion capacity assessment, plethysmography, and lung clearance index (LCI), high-speed video microscopy, gastric aspirate sampling, and Flexible fiberoptic bronchoscopy (FFB) during clinical follow-up, and hospitalization numbers of patients with chronic lung diseases during the COVID-19 pandemic and compared them with the previous year before the pandemic started.
RESULTS
In 2020, compared with 2019, outpatient visits decreased by 42.2%; from 8324 patients to 4804 patients and other laboratory procedures decreased after the pandemic started; PFTs including spirometry by 87.2%; from 2990 to 380 tests, diffusion capacity assessment from 172 to 55 tests, plethysmography from 172 to 53 and LCI from 70 to 9 tests, also high-speed video microscopy analyses by 64.3% from 300 to 107 patients, and gastric aspirate sampling by 75.5% from 42 to 9 patients. FFB procedures also decreased by 59.1%, from 142 to 58 patients, and only 15 were after the first reported case of COVID-19 in our country, and they were all for urgent indications. None of these patients was positive pretesting for COVID-19 who underwent the FFB. There was no significant decrease in patients' hospitalization due to pulmonary exacerbation in cystic fibrosis, interstitial lung disease, and primary ciliary dyskinesia.
Topics: Adaptor Proteins, Signal Transducing; Adolescent; COVID-19; Child; Child, Preschool; Cystic Fibrosis; Delivery of Health Care; Humans; Infant; Pandemics; Pulmonary Medicine; SARS-CoV-2
PubMed: 34265185
DOI: 10.1002/ppul.25557 -
HardwareX Apr 2022Nanoscale positioning has numerous applications in both academia and industry. A growing number of applications require devices with long working distances and nanoscale...
Nanoscale positioning has numerous applications in both academia and industry. A growing number of applications require devices with long working distances and nanoscale resolutions. Friction-inertia piezoelectric positioners, which are based on the stick-slip mechanism, achieve both nanometer resolution and centimeter-scale travel. However, the requirements of complex preload mechanism, precision machining, and precise assembly increase the cost of conventional friction-inertia nanopositioners. Herein we present the design of an open-source -axis nanopositioning system. Utilizing a magnet-based stick-slip driving mechanism, the proposed nanopositioner provides several advantages, including sub-nanometer resolution, a payload capacity of up to 12 kg (horizontal), compact size, low cost, and easy assembly; furthermore, the system is adjustment-free. The performance tests validate the precision of the system in both scanning and stepping operation modes. Moreover, the resonant spectra affirm the rigidity and dynamic response of the mechanism. In addition, we demonstrate the practical applications of this nanopositioner in various measurement techniques, including scanning electron microscopy, vibrometry, and atomic force microscopy. Furthermore, we present 11 variations of the nanopositioner designs that are either compatible with ultra-high-vacuum systems and other existing systems, 3D printable, or hacking commercial linear slides.
PubMed: 35647417
DOI: 10.1016/j.ohx.2022.e00317 -
Scientific Reports Jul 2022Total holographic characterization (THC) is presented here as an efficient, automated, label-free method of accurately identifying cell viability. THC is a...
Total holographic characterization (THC) is presented here as an efficient, automated, label-free method of accurately identifying cell viability. THC is a single-particle characterization technology that determines the size and index of refraction of individual particles using the Lorenz-Mie theory of light scattering. Although assessment of cell viability is a challenge in many applications, including biologics manufacturing, traditional approaches often include unreliable labeling with dyes and/or time consuming methods of manually counting cells. In this work we measured the viability of Saccharomyces cerevisiae yeast in the presence of various concentrations of isopropanol as a function of time. All THC measurements were performed in the native environment of the sample with no dilution or addition of labels. Holographic measurements were made with an in-line holographic microscope using a 40[Formula: see text] objective lens with plane wave illumination. We compared our results with THC to manual counting of living and dead cells as distinguished with trypan blue dye. Our findings demonstrate that THC can effectively distinguish living and dead yeast cells by the index of refraction of individual cells.
Topics: Coloring Agents; Holography; Microscopy; Microscopy, Video; Saccharomyces cerevisiae
PubMed: 35882977
DOI: 10.1038/s41598-022-17098-y -
Cancers Nov 2023Actinic keratosis (AK), due to its widespread prevalence, as well as the possibility of progression to an invasive form of squamous cell carcinoma, requires treatment...
Actinic keratosis (AK), due to its widespread prevalence, as well as the possibility of progression to an invasive form of squamous cell carcinoma, requires treatment regardless of the clinical stage. New imaging techniques, such as in vivo reflectance confocal microscopy (RCM), significantly increase the accuracy of diagnosis and allow noninvasive evaluation of the therapeutic efficacy of the ongoing treatment. Our objective was to evaluate the prevalence of specific (video)dermoscopy and RCM features of pigmented and classical subtypes of AK before and after photodynamic therapy (PDT) treatment. We included patients with facial grade II AKs (25 pigmented, 275 non-pigmented) were included in the study. Skin lesions were evaluated by (video)dermoscopy and RCM at the baseline and three months after PDT. In classic AK, the most frequent dermoscopic findings were fine wavy vessels (96%), scale (92%), microerosions (48%), and "strawberry" pattern (36%), while pigmented AK was characterized mostly by "rhomboidal pattern" (80%), scale (60%), white globules (48%), "jelly sign", and superficial pigmentation (40%). RCM's most characteristic classic AK findings were abnormal honeycomb pattern in the spinous layer, epidermal inflammatory infiltrate, and solar elastosis that were present in 96% of lesions. Pigmented AKs presented mostly with dark central areas of parakeratosis (72%), mottled pigmentation (72%), dermal inflammatory infiltrate (64%), solar elastosis (60%), and abnormal honeycomb pattern in the spinous layer (56%). Dermoscopically, PDT resulted in complete disappearance of the "rhomboidal pattern" in both classical and pigmented AKs, "starburst pattern" and "jelly sign" in classical AKs, and inner gray halo, "rosette sign" and central crust in pigmented AKs. Three months after one PDT session, RCM evaluation showed mostly solar elastosis in both classical and pigmented AK subtypes, epidermal inflammatory infiltrate in classical AKs, and dermal inflammatory infiltrate in pigmented AKs. New noninvasive imaging techniques such as RCM and (video)dermoscopy can help practitioners better visualize the efficacy of the ongoing PDT treatment in either classical or pigmented AK subtypes.
PubMed: 38067302
DOI: 10.3390/cancers15235598